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Identification of an Autoimmune Serum Containing Antibodies Against the Barr Body

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Identification of an Autoimmune Serum Containing Antibodies Against the Barr Body Identification of an autoimmune serum containing antibodies against the Barr body Bo Hong*, Peter Reeves†, Barbara Panning†, Maurice S. Swanson‡§, and Thomas P. Yang*§¶ʈ *Department of Biochemistry and Molecular Biology, ‡Department of Molecular Genetics and Microbiology, §Center for Mammalian Genetics, and ¶Division of Pediatric Genetics, University of Florida College of Medicine, Gainesville, FL 32610; and †Department of Biochemistry and Biophysics, University of California, San Francisco, CA 94140 Communicated by Stanley M. Gartler, University of Washington, Seattle, WA, May 24, 2001 (received for review April 20, 2001) Transcriptional inactivation of one X chromosome in mammalian components and structure of the Barr body itself. The Barr body female somatic cells leads to condensation of the inactive X has been examined by electron microscopy (6, 7), and the results chromosome into the heterochromatic sex chromatin, or Barr body. indicate the possibility of a special nuclear envelope attachment Little is known about the molecular composition and structure of region for the Barr body. But these studies provide few insights the Barr body or the mechanisms leading to its formation in female into the possible composition or macromolecular organization of nuclei. Because human sera from patients with autoimmune dis- the inactive X chromosome. The Barr body and individual genes eases often contain antibodies against a variety of cellular com- on the inactive X have been probed with nucleases, particularly ponents, we reasoned that some autoimmune sera may contain DNase I, to analyze molecular structure. Nick translation assays antibodies against proteins associated with the Barr body. There- on female cells after fixation and nicking with DNase I have fore, we screened autoimmune sera by immunofluorescence of shown that inactive X chromatin is resistant to nick translation human fibroblasts and identified one serum that immunostained a (6, 8). However, analysis of general DNase I sensitivity of the distinct nuclear structure with a size and nuclear localization X-linked mouse Hprt and human Pgk genes in unfixed cells (9, consistent with the Barr body. The number of these structures was 10) showed a much smaller difference in sensitivity between the consistent with the number of Barr bodies expected in diploid active and inactive alleles (Յ2-fold) than would be expected for female fibroblasts containing two to five X chromosomes. Immu- highly condensed heterochromatin (i.e., inactive X) versus un- nostaining with the serum followed by fluorescence in situ hybrid- condensed euchromatin (i.e., active X). Recent analysis of the ization with a probe against XIST RNA demonstrated that the major three-dimensional organization of the active and inactive X fluorescent signal from the autoantibody colocalized with XIST chromosomes showed the two chromosomes occupy the same RNA. Further analysis of the serum showed that it stains human volume, but the active X appeared flatter with a larger and metaphase chromosomes and a nuclear structure consistent with fuzzier surface than the inactive X, which appeared rounder in the inactive X in female mouse fibroblasts. However, it does not shape with smoother surface structure (11). Two studies have exhibit localization to a Barr body-like structure in female mouse also examined the potential association of the two telomeres and embryonic stem cells or in cells from female mouse E7.5 embryos. resulting loop structure of the inactive X chromosome (12, 13). The lack of staining of the inactive X in cells from female E7.5 Currently, three macromolecules have been shown to colo- embryos suggests the antigen(s) may be involved in X inactivation calize with the Barr body or inactive X chromosome. at a stage subsequent to initiation of X inactivation. This demon- Perichromin, a nuclear envelope protein directly or indirectly stration of an autoantibody recognizing an antigen(s) associated bound to DNA (14), has been reported to be associated with the with the Barr body presents a strategy for identifying molecular Barr body (15). However, its role in X inactivation, if any, is components of the Barr body and examining the molecular basis of unknown. The XIST gene encodes a large nuclear RNA found X inactivation. to be associated exclusively with the inactive X chromosome by fluorescence in situ hybridization (FISH) (16, 17). This RNA uring early mammalian female embryogenesis, one of the exhibits no conserved and extended ORF (16, 18), is transcribed Dtwo transcriptionally active X chromosomes is inactivated in only from the inactive X chromosome (19, 20), and is essential each cell of the embryo (1). The stable inactivation of genes on for normal X inactivation (21, 22). The mechanisms by which the one of the two X chromosomes in females functionally equalizes XIST gene and RNA function in X inactivation are currently the apparent dosage imbalance of X-linked genes between males unresolved. The histone variant macroH2A1.2 also is reported to and females. This chromosome-wide transcriptional silencing is be concentrated on the inactive X chromosome (23, 24), al- associated with condensation of the inactive X chromosome into though association of macroH2A1.2 with the inactive X does not the heterochromatic sex chromatin or Barr body, a unique appear to be required for either initiating or maintaining tran- constituent of the female nucleus identified half a century ago scriptional repression of genes on the inactive X chromosome et al. (2). The Barr body in female interphase nuclei is characteristi- (25, 26). Recently, Perche have reported that, in addition to macroH2A, the core histones H2B and H3 also show pref- cally found as a darkly staining nuclear inclusion commonly erential localization to the Barr body, suggesting that the Barr associated with the nuclear membrane (2). In cells carrying a body may contain a higher density of nucleosomes (27). Con- diploid complement of autosomes, X inactivation and Barr body versely, the inactive X chromosome is reported to be deficient in formation occurs according to the N-1 rule: cells maintain a another novel histone variant, termed H2A-Bbd (28). Despite single active X chromosome and inactivate and condense all these promising findings, knowledge of the composition and remaining X chromosomes (3, 4). However, Barr body formation molecular structure of the Barr body, as well as mechanisms of does not appear to be a requirement for maintaining transcrip- global silencing of genes on the inactive X, remains incomplete. tional repression of genes on the inactive X because rodent– human somatic cell hybrids containing an inactive human X chromosome do not form Barr bodies but continue to maintain Abbreviations: FISH, fluorescence in situ hybridization; ES, embryonic stem. transcriptional silencing of genes on the inactive human X (5). ʈTo whom reprint requests should be addressed. E-mail: [email protected]fl.edu. The molecular mechanisms for establishing and maintaining The publication costs of this article were defrayed in part by page charge payment. This GENETICS this unique system of differential gene regulation are not well article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. understood, and currently little is known about the molecular §1734 solely to indicate this fact. www.pnas.org͞cgi͞doi͞10.1073͞pnas.151259598 PNAS ͉ July 17, 2001 ͉ vol. 98 ͉ no. 15 ͉ 8703–8708 Downloaded by guest on September 28, 2021 Fig. 1. Detection of Barr bodies by autoimmune serum 154. Indirect immunofluorescence was performed on diploid human fibroblasts containing different numbers of X chromosomes using autoimmune serum 154 (1:200 dilution) and an FITC-conjugated secondary antibody against human immunoglobulins. Images were collected with a Bio-Rad 1024 ES confocal microscope. (A) GM00468 (46,XY). (B) GM06111 (46,XX). (C) GM00254 (47,XXX). (D) GM01415E (48,XXXX). (E) GM05009C (49,XXXXX). (F) Human-hamster hybrid cell line 8121 (containing an inactive human X chromosome). Arrows indicate Barr bodies. Antisera from human patients with autoimmune diseases have bryonic fibroblasts in medium containing leukemia inhibitory been used extensively as a tool for studying intracellular struc- factor. Female mouse fibroblasts containing three X chromo- ture and function (29, 30). We reasoned that a small subpopu- somes were a gift of Catherine Brisken (Whitehead Institute). lation of autoimmune patients may carry antibodies against one or more components of the Barr body. Further, autoantisera Isolation of Mouse Embryos. Cells from E7.5 embryos were isolated with antibodies against the Barr body then could be used to as described (32). identify the corresponding Barr body-associated antigen. There- fore, to examine whether or not human autoimmune sera may be Indirect Immunofluorescence. Human fibroblast cells were grown useful as probes of Barr body composition and structure, we overnight on glass microscope slides and fixed with 2% form- screened samples of autoantisera by an indirect immunofluo- aldehyde for 15 min at room temperature. Fixed cells were rescence assay on male and female fibroblasts and examined the treated with acetone at Ϫ20°C for 5 min, then at room temper- ͞ immunostaining patterns for a female-specific staining pattern ature blocked with 3% BSA in PBS (2 mM KH2PO4 8mM ͞ ͞ and colocalization of antibody with the Barr body. Screening of Na2HPO4 2.5 mM KCl 140 mM NaCl, pH 7.2) for 15 min, 255 different autoimmune sera identified one serum containing incubated with diluted autoantiserum for 1 h, washed with PBS, antibodies against the Barr body in both human and mouse incubated with FITC-conjugated goat anti-human Ig for 1 h, fibroblasts. washed again with PBS, counterstained with 1 ␮g͞ml DAPI in PBS for 10 min, mounted in 1 mg/ml p-phenylenediamine͞10 Materials and Methods mM Tris⅐HCl, pH 8.5͞90% glycerol, and examined with an Autoimmune Sera. Sera were obtained from patients with auto- Olympus fluorescence microscope. Images in Figs. 1 and 3 were immune diseases including systemic lupus erythematosus, sclero- collected with a Bio-Rad 1024-ES confocal microscope.
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